CH641570A5 - Process for the determination of an immunologically active material and reagent for carrying out this process - Google Patents

Description

The invention relates to a method for determining immunologically active materials and to a reagent for performing this method.

40 In recent years, numerous analytical systems based on competing protein binding analysis (also known as saturation analysis) have been developed.

The terms “saturation analysis” and “competing protein formation analysis”, which are synonymous, refer to analytical systems that are used to determine immunologically active materials (ligands). The results of these determinations in biological fluids are used in medical and veterinary diagnosis. The diagnosis depends on whether the content of the particular substance is normal or pathological. The analytical principle is based e.g. on competition between a ligand and a labeled ligand for a common specific binding agent (receptor), as illustrated by the following equation:

55

Labeling ligand + ligand + receptor— »•

6o marking marking

Ligand + ligand + ligand + ligand

I I

Receptor Receptor 05 (1) (2) (3) (4)

Equation 1

The primary reaction is a combination of a ligand molecule with a receptor molecule to form one

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bimolecular ligand-receptor complex (1). A secondary reaction is caused by the addition of a labeled ligand, which similarly combines with the receptor to form a labeled ligand-receptor complex (2). Concentrations of the receptor and the labeled ligand are constant in the saturation analysis. The receptor concentration is limited so that the labeled ligand is in excess relative to the receptor. Under these conditions, the addition of the ligand triggers competition between the ligand and the labeled ligand when binding to the receptor. Consequently, increasing the ligand concentration decreases the amount of labeled ligand-receptor complex. The test principle is based on the determination of the percentage of the total labeled ligand bound to the receptor. This percentage is inversely proportional to the amount of reaction mixture from the sample to be measured or from the ligand used in the standard used in the test. The decrease in the concentration of the labeled ligand-receptor complex or the increase in the concentration of the labeled ligand in the reaction mixture can be used to determine the ligand concentration.

The sensitivity of the saturation analysis depends on the use of a receptor that has a very high affinity for the ligand and for the labeled ligand. On the other hand, the sensitivity also depends on the use of a label that can be detected at a very low concentration.

The specificity of the saturation analysis depends on the ability of the receptor to bind only the ligand and the labeled ligand in a complex mixture of different molecules.

Saturation analysis has been used using a variety of different techniques, the differences being primarily related to the type of label used. In general, these techniques are categorized by the broad terms "radioactive test" or "non-radioactive test" depending on whether or not a radioactive tracer is used as a label.

The radioactive test has been used to a greater extent than non-radioactive tests. The radioactive test can be further classified as a radioimmunoassay or a radio receptor test, depending on the type of receptor used in the test. The radioimmunoassay uses an antibody that specifically binds the ligand and the labeled ligand. On the other hand, radioreceptor analysis refers to the use of any other type of biological receptor that similarly specifically binds the ligand and the labeled ligand.

In all radioactive test techniques, it is essential to physically separate the bound fraction (1 and 2 in Equation 1) from the unbound fraction (3 and 4 in Equation 1) of the reaction mixture. An index of ligand concentration can then be obtained by counting these fractions in a radioactivity counter and comparing the counts obtained for unknown samples with those obtained for suitable standard ligand samples subjected to the same test. Numerous different and divergent methods have been described for the separation of the bound and free portions of the radioactive test reaction mixture,

techniques such as Gel filtration, absorption and ion exchange chromatography, fractional precipitation, solid phase or electrophoresis, were used.

After the development of radioactive test techniques in saturation analysis, methods were developed that use non-radioactive labels. Procedure under

The use of enzymes as a label has been shown. These methods can have the advantage that physical separation of the bound (1 +2) and free (3 + 4) fractions or portions of the labeled ligand is not necessary during the test procedure.

When an antibody binds to a ligand labeled with an enzyme, the activity of the enzyme is changed. The degree or extent of the change in enzyme activity is an indication of the concentration of the labeled ligand in the bound fraction and consequently provides a concentration index of the ligand in the reaction mixture.

The chemical structure of the ligand-enzyme complex is extremely difficult to determine, and this is a major disadvantage of the enzyme immunoassay. This is undoubtedly due to the large variety of amino acid side chains that are available on the enzyme surface for complex formation with the ligand. This leads to great difficulties in reproducing the ligand enzyme in various preparations of the complex. The general lack of control over the complexation reaction results in the binding of many ligand molecules to a single enzyme molecule, although it may be easy for antibodies to inhibit enzyme activity by binding only a few of these ligand molecules. Therefore, not all antibody-labeled antigen interactions lead to a change in enzyme activity, which reduces the sensitivity of this technique.

Protein-protein interaction between different antibody molecules and antibody and enzyme molecules is another consequence of the diversity of the ligand molecules on the enzyme surface. Protein-protein interaction also increases when the ligand is also a polypeptide. The enzyme molecule thus induces a localized microenvironment of high protein concentration. In such situations, it has been shown that protein precipitation occurs, causing the loss of one of the main advantages of the enzyme immunoassay by requiring the separation of antibody-bound and free fractions.

A modification of the enzyme immunoassay has been described which partially overcomes these problems by labeling the ligand with a low molecular weight detector molecule. In this test, the antibody binding of the labeled ligand sterically prevents the binding of one detector molecule by another antibody specific for the detector molecule. The extent of the hindrance is determined by the competition for the binding of the free ligand-detector molecule and the enzyme labeled with the detector molecule with the detector molecule antibody. The degree of change in enzyme activity, as determined in the normal enzyme immunoassay, gives an indication of the concentration of free ligand detector molecule, which in turn indicates the concentration of the ligand in the reaction mixture.

The advantage of this technique is that a small molecule is bound to the ligand instead of an enzyme, which allows the chemical structure of the labeled ligand to be determined, thereby overcoming many of the disadvantages of the enzyme immunoassay previously described. However, this system only overcomes the disadvantages of the primary binding reaction involving the ligand and the labeled ligand and transfers them to the detector system to determine the extent of the antibody-bound and antibody-free portions of the labeled ligand.

The disadvantages of the methods according to the prior art are overcome by the invention, according to which an enzyme modification is used as the label.

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The invention relates to a method for determining an immunologically active material in a sample, the sample being labeled with a receptor which is capable of specifically binding the immunologically active material, the immunologically active material which is labeled with a substance which is capable of modifying the activity of an enzyme , an enzyme and an enzyme substrate, and the degree or type of enzyme activity obtained is measured and compared with that obtained with a standard.

One embodiment of the method according to the invention is that the immunologically active material is brought together in an insolubilized form with an enzyme and an enzyme substrate, the degree or type of enzyme activity being measured after the solid phase has been separated off and compared with that obtained with a standard .

The invention further relates to a reagent for carrying out the method, which is able to bind the immunologically active material specifically, labeled with a substance which is able to modify the degree or the type of activity of an enzyme.

The terms "immunologically active material" or "ligand" in this specification refer to any or part of an immunologically active substance that is immunologically determinable, e.g. using techniques of saturation analysis. An essential requirement is that there is a receptor that specifically binds the ligand. If the receptor is an antibody, the ligand is a hapten or antigen, so that a specific antibody can arise. A ligand is called a hapten if it only produces antibodies when bound to a compound with antigenic properties. On the other hand, a ligand is called an antigen if it shows antibody formation without chemical modification. The ligand can vary widely in molecular weight, from about 100 to 1,000,000. This molecular weight range does not set any limits in the test, provided that a receptor is available which specifically binds the ligand.

The ligand can be structurally either polymeric or non-polymeric. If polymeric, it will usually be of biological origin and classified as a nucleic acid polysaccharide and / or polypeptide. On the other hand, if the ligand is non-polymeric, it generally has a molecular weight of less than 2000 and can have numerous structures, functionalities and physiological properties.

Particularly important ligands are amines, amino acids, peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, mono- and polysaccharides, alkaloids, vitamins, drugs, narcotics, antibiotics, metabolites, pesticides, toxins, industrial contaminants, taste and Flavoring agents, hormones, enzymes, coenzymes, cellular or extracellular tissue components and antibodies isolated from humans or animals. However, the test is not limited to these ligands only.

Components with an immediate possibility for analysis with the system are hepatitis B surface antigen, ferritin, tumor antigens such as CEA, a-fetoprotein, rheumatoid factor, C-reactive proteins, immunoglobulin classes IgG, IgM or IgA, myoglobulin , Thyroid hormones including T3 and T », insulin, steroid hormones, including testosterone or estradiol, abuse-prone medicines, such as narcotic analgesics such as morphine, barbiturates, stimulants, such as amphetamine, medicines for the treatment of epilepsy, including diphenylhydrogenoin and phenobarco-glycerin, and cardiac glycosylamine like digoxin, vitamins like vitamin Bj2 and folic acid. Antibodies that are directly accessible for analysis with the system are also antibodies that are available with a

Infection associated with syphillis, gonorrhea, brucellosis, rubella and rheumatism.

The term “labeled immunologically active material” or “labeled receptor” in the present description refers to a ligand, an analogue of a ligand or its part or a receptor that is labeled with an enzyme modifier. One, more than one and generally less than 100 labeling molecule (s) can be attached to a ligand or a receptor molecule. Similarly, one, more than one and usually less than 5 ligand or receptor molecule (s) may be attached to a label molecule. Linking separate labeling molecules to a ligand or receptor molecule generally increases the sensitivity of the test, provided the special labels do not interfere with binding.

Linking a modifying molecule to a ligand or receptor molecule involves the formation of intermolecular bonds that are mostly, but not necessarily, covalent in nature. The linkage can sometimes take place in the presence of a coupling agent by inserting a connecting group between the label and the ligand or the receptor.

The modifying molecule can be linked directly to the ligand or the receptor molecule. However, it may be desirable to incorporate chemical bridges of different lengths between the modifying molecule and the ligand or receptor molecules, depending on the particular test envisaged. Sometimes it may even be advantageous to bind the modifying molecule and the ligand or receptor molecules separately to the same carrier molecule, e.g. a macromolecule, such as a polypeptide or a polysaccharide.

The term “receptor” used here refers to any substance that is capable of specifically binding a ligand or a labeled ligand or a part thereof. In general, the receptor used in the test is a specific antibody to the ligand formed in the blood of vertebrates after the injection of a suitable hapten or antigen. On the other hand, naturally occurring receptors can also be used in the test. This latter group includes, but is not limited to, proteins, nucleic acids, and cell membranes. Such receptors have been used in radioactive test techniques for thyroxine, insulin, angiotensin and various steroid hormones.

In the event that the ligand is an antibody, the receptor may be the antigen used to induce the antibody in a host animal. In another embodiment, the receptor can be an antibody against the antibody to be determined.

It is impossible to determine with certainty the type of receptor action which, when the labeled ligand binds, reduces the interaction between the enzyme and the modifying molecule. The most plausible explanation is that the affinity of the enzyme for the modifying molecule is reduced as a result of a change in the size and charge of the complex receptor-ligand-modifier compared to the ligand-modifier alone.

The term “modifying substance” or “modifier” in the present description refers to any substance that is able to interact with an enzyme in such a way that the extent or the degree or the type of enzyme activity is modified or modified. This modification can lead to inhibition, activation or a change in the specificity or any other property of the enzyme which is detectable either directly or indirectly by a change in the enzyme activities or in the type of activity, e.g. a change in

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Reaction conditions such as cofactor requirements or pH optimum, the kinetic properties or the activation energy. The modifying substance can range in size from a small molecule to a macromolecule, and its interaction with an enzyme molecule can be either reversible or irreversible, depending on whether the intermolecular association is ionic or covalent in nature.

The sensitivity of the test depends, among other things. depends on the affinity of the receptor for the ligand and the ability of the modifying substance to cause a change in the degree of the type of enzyme activity. The extent or type of enzyme activity is preferably modified with a minimum concentration of modifying substance. The closer this concentration is to that of the enzyme on a molecular basis, the greater the sensitivity of the test.

The modifying substance is preferably an enzyme inhibitor which, when interacting with an enzyme, inhibits its activity. The mode of action of the inhibitor can be competitive, non-competitive, non-competitive and / or all-steric. Preferably, the inhibitor or inhibitor should have an inhibition constant (inhibitor concentration necessary for 50% inhibition of the enzyme system) below 10 -3 mol / 1, depending on the test performed. The inhibition constant is particularly preferably between 10 ~ 15 and 10 "5.

Any enzyme can be used in the assay, provided there is a modifying agent that specifically modifies enzyme activity in the manner described above. Enzymes of choice are stable, easily available at low cost, have a high number of changes and a simple test system. The number of changes (product molecules formed in 1 min per enzyme molecule) is preferably over 100, depending on the test carried out specifically. The number of changes is particularly preferably as high as possible and at least 200.

Enzyme-modifying systems which are particularly useful according to the invention are dihydrofolate reductase / methotrexate, dihydrofolate reductase / 4-aminopterin, dihydrofolate reductase / other specific inhibitors of this enzyme, β-glucuronidase / 4-deoxy-5-amino-sugar acid and their derivatives, biotin-containing enzymes / avidin, such as carboxylase / avidin, chymotrypsin / TPCK

S0o-HH-C-C -CH «C1

CE

y-cystathionase / propargylglycine, alanine racemase / tri-fluoroalanine, tryptophanase / trifluoralanine, tryptophan synthetase / trifluoralanine, ß-cystathionase / trifluoralanine, pyruvate-glutamate-transaminase / trifluoroacid-oxidase, 3-lactic acid oxidase, 3-lactic acid oxidase, 2-lactic acid oxidase, 3-lactic acid oxidase, 2-lactic acid oxidase, 2-lactic acid oxidase, 2-lactic acid oxidase, 2-lactic acid oxidase, Monoamine oxidase / N, N-dimethyl-propargylamine and diamine oxidase / H2N-CH-C = C-CH2-NH2.

The enzyme activities can be determined by direct or indirect recording of substrate consumption or product formation at a suitable pH and temperature using detection systems such as colorimetry, spectrophotometry, fluorescence spectrophotometry, gasometry, thermometry (toning), scintillation counting.

To increase the sensitivity of the system, bioluminescence and enzyme cycle techniques can be used, e.g. the techniques as described by J. Lee et al. in Liquid Scintillation Counting: Recent Developments, Stanley P.E. and Scoggins, B.A., Academic Press, New York, p. 403 and Lowry O.H. et al. in J. Biol. Chem. 236. pp. 2746-2755.

In one embodiment of the method according to the invention, a labeled ligand can be used to determine the presence of a ligand in an unknown sample by simultaneously or successively adding the labeled ligand and the unknown sample to an aqueous medium at a suitable pH and with a content of for the ligand and the labeled one Ligand specific receptor can be used. After a suitable incubation period, the distribution of the receptor bound to the ligand and the labeled ligand is determined by adding enzyme and substrates. The receptor, which is specific for the ligand, also binds the labeled ligand, thereby reducing the interaction between the modifying agent and the enzyme and thus reducing the variation in enzyme activity. The addition of the ligand in the test competes with the labeled ligand in binding to the receptor and thereby reduces the concentration of free ligand in the sample. The interaction between ligand / modifying agent and enzyme increases and the enzyme activity is in turn impaired. The change in enzyme activity is therefore a function of the ligand concentration in the sample and is caused by unbound labeled ligands. Thus, the difference between the enzyme activity obtained and the activity obtained in the absence of the ligand is an indication of the concentration of the ligand in the unknown sample.

One of the main advantages of this process is

that a separation of bound and free shares in the way of working is unnecessary. However, this does not exclude the use of such a separation step in the test after the incubation of ligand and labeled ligand with the receptor and before the estimation of the enzyme activity. Separation of the fractions or fractions may sometimes be desirable in order to remove substances in the sample which can interfere with the enzyme test. The separation can be performed using any of the many techniques described for the radioimmunoassay, including gel filtration, adsorption and ion exchange chromatography, fractional precipitation, solid phase, and electrophoresis. Of course, this test is not limited to the determination of haptens and antigens as ligands, but can also be adapted to identify and measure antibodies as ligands.

This can e.g. by labeling the antibody with an enzyme modifier and measuring the degree of change in enzyme activity after incubation of the labeled antibody and the antibody of the sample with a limited concentration of antigen or hapten. The change or change in enzyme activity is related to the concentration of the antibody in the sample. If necessary, the bound and free portions can be separated before adding the enzyme and the substrate.

The method according to the invention also makes it possible to use a labeled receptor instead of a labeled ligand to determine a ligand. A ligand in the unknown sample reacts with excess receptor labeled with an enzyme modifying agent and, after incubation, excess insolubilized solid phase ligand is added and reacts with the remaining free labeled receptor. After separation of the solid phase, the change in enzyme activity, the

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is connected to the soluble ligand, measured and based on the ligand concentration.

The required reagents can also be used in a "sandwich" technique, provided that the ligand has at least two attachment points. The ligand reacts with excess solid phase receptor, and after incubation with subsequent washing, the solid phase receptor / ligand is reacted with excess receptor that is labeled with an enzyme-modifying agent. Free labeled receptor is removed by washing and the extent of enzyme change in the separated fractions is determined. This then gives an index of the ligand concentration.

The following examples illustrate the invention.

example 1

Production of «Amino digoxin»

10 ml of 0.2 M sodium metaperiodate were added to a suspension of 156 mg (0.2 mmol) of digoxin in 5 ml of absolute ethanol while stirring. The mixture became homogeneous after 10 minutes and then a precipitate slowly formed. After 2 hours, 5 ml of water and 5 ml of ethanol were added. 122 ml (2.2 mmol) of ethylene glycol was added after a further 30 min and a dense white precipitate began to form immediately. After stirring for 80 min, 133 ml of (2.0 mmol) ethylenediamine was added and the resulting pH of 11.0 was adjusted to 9.5 with 0.1 M HCl and the reaction mixture was allowed to stand at room temperature for 18 h. The pH did not change during this time.

151.4 mg (4.0 mmol) of sodium borohydride was then added and the mixture was stirred for 3.5 h. The pH was then adjusted from 10.5 to 6.5 with 1 M formic acid (about 3 ml) and a thin layer chromatogram of this mixture showed a single main spot with an Rf value of 0.15 (silica gel on aluminum, developed in butanol / Acetic acid / water 4: 1: 1). Digoxin had an Rf of 0.7 when developed in the same system.

The solvents were evaporated to dryness under vacuum in a rotary evaporator with a water bath at 60 °. The last few ml of water were removed by adding 95% ethanol (3 x 20 ml) and repeating the above evaporation.

The resulting pale yellow solid was extracted three times with absolute ethanol and these combined extracts were concentrated to about 4 ml and centrifuged to separate a small amount of salt which was discarded. The pale yellow supernatant solution was evaporated to dryness under a stream of dry nitrogen to give a yellow oil which showed the same Rf on thin layer chromatography as the reaction mixture described above. The product was found to contain a free amino group by reacting it with fluram (4-phenylspiro [fluran-2 (3H), l'-phthalan] -3,3'-dione) to form an intensely fluorescent compound. The product showed a spectrum in concentrated sulfuric acid similar to that of digoxin, with absorption peaks at 385 and 495 nm (m | i). The product also showed a strong affinity for antisera (rabbits) that are specific for digoxin.

Example 2

Preparation of methotrexate-aminodigoxin conjugate

11 mg of methotrexate were dissolved in 5 ml of H2O and adjusted to pH 6.5. 10 mg "aminodigoxin" was dissolved in this solution and the volume was adjusted to 20 ml with H2O. The pH was again adjusted to 6.5 and 484 mg of N-ethyl-N "- (3-dimethylamino) propyl-carbodiimide-

Hydrochloride dissolved in 5 ml of H2O was added to the reaction mixture. The pH was kept at 6.2 at room temperature for 24 h. The conjugal was purified on a silica gel column using 3% ammonium citrate as the solvent. The fractions containing the desired product were combined and showed the double ability to bind antisera (rabbits), which are specific for digoxin, strongly and also to strongly inhibit the enzyme dihydrofolate reductase (chicken liver).

Example 3

Enzyme inhibitor immunoassay for digoxin

100 ul of serum were incubated for 15 min at 30 ° C. with 100 ul of anti-goxin antibody solution, 100 ml of NADPH solution, 100 ml of 2-mercapto-ethanol solution and 550 ml of sodium phosphate buffer pH 7.5 j-il methotrexate-digoxin conjugate of Example 2 (70 | i, g / ml) was added, and the mixture was incubated for 15 min, after which 100 ml of dihydrofo-lat reductase solution were added, the dihydrofolate reductase used Preparation was isolated from chicken liver according to the method of Kaufman, BT, & Gardiner, RC, Journal of Biological Chemistry, vol. 211, p. 1319 (1966) .The mixture was incubated for a further 3 min and the enzyme activity by adding 100 ml The dihydrofolate solution was determined and recorded at 340 nm with a Varian Schreberber spectrophotometer, and the results are shown in the following table.

Table I

Reaction components enzyme activity

Digoxin

anti-

Metho-

Enzyme-

OD / min

%

(Samples digoxin

trexat dig

test-

Hem conc

Antibody

oxine

Compo-

mation tration per

Conjugates

ng / ml)

(ng in test sample)

0

is missing

0

available

0.150

0

0

is missing

7

available

0.100

33

0

available

7

available

0.145

3rd

5

available

7

available

0.125

16

10th

available

7

available

0.115

23

The reaction components were added in the order described above.

OD = optical density

The above results indicate that a few ng digoxin in serum can be determined in the described system.

Example 4

Preparation of a conjugate from methotrexate and human serum albumin

45 mg of methotrexate was dissolved in 1.0 ml of N, N-dimethylformamide and 25 mg of N-hydroxysuccinimide were added.

41 mg of N, N-dicyclohexylcarbodiimide were dissolved and the mixture was kept at room temperature for 13 hours. The insoluble urea by-product was filtered off and 100 ml of the filtrate was added to a solution containing 5 mg of human serum albumin in 800 ml of 0.1 M sodium phosphate buffer of pH 7.5 and 100 µl of dioxane

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The reaction mixture was kept at room temperature for 30 minutes and then placed on a Sephadex G-25 column which was equilibrated with 0.2 M sodium phosphate buffer of pH 7.5. The column was developed with this buffer and two elution peaks containing methotrexate were obtained, the first of which contained the conjugate of methotrexate and human serum albumin.

Example 5

Enzyme inhibitor immunoassay for human serum albumin 100 | il diluted serum were at 30 ° C for 15 min with 100 U.1 anti-human serum albumin antibody (rabbit) solution, 100 ni NADPH solution, 100 u.12 mercaptoethanol solution and 550 ml of pH 7.5 sodium phosphate buffer. 100 μl methotrexate-human serum albumin conjugate (8 μg / ml) was added and the mixture was incubated for 15 min, after which 100 μl dihydrofolate reductase solution was added. The mixture was incubated for a further 3 min and the enzyme activity was determined by adding 100 μl of dihydrofolate solution and recorded at 340 nm using a Varian-Schreiber spectrophotometer. The results are shown in Table II below.

Table II

Reaction components

Enzyme activity

Human

anti-

Metho-

Enzyme-

OD / min

%

serum human

trexate

test-

Hem albumin serum

Human ser

Compo-

mung

(Samples of albumin

around-

nenten

conc.

anti-

albumin-

Hg / ml)

body

Conjugate (Hg in test sample)

0

is missing

0

available

0.123

0

0

is missing

0.8

available

0.068

45

0

available

0.8

available

0.105

16

5

available

0.8

available

0.092

25th

10th

available

0.8

available

0.085

31

The above results clearly show that a few human serum albumin can be determined in the system described.

Example 6

Synthesis of methotrexate-aminoethylmorphine conjugate a) Synthesis of 03-aminoethylmorphine

400 mg of LAH were suspended under nitrogen in 10 ml of tetrahydrofuran (THF) freshly distilled from lithium aluminum hydride (LAH). A solution of 400 mg of morphine and 400 mg of chloroacetonitrile in 4 ml of freshly distilled THF was added over 5 minutes, followed by refluxing for 1 hour. The mixture was allowed to cool and 0.6 ml of water was added, then 0.6 ml of 10 weight percent sodium hydroxide and 2 ml of water. After filtering the mixture, the salts were washed with THF, the THF portions were combined, dried with magnesium sulfate under nitrogen, filtered and the filtrate evaporated to give 380 mg of 03-aminoethylmorphine.

b) Synthesis of N-t-butoxycarbonyl-y- (03-aminoethylmorphine) -glutamic acid a-benzyl ester

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A mixture of 03-aminoethylmorphine (50 mg, prepared as above), N-t-BOC-glutamic acid a-benzyl ester (34 mg) and dicyclohexylcarbodiimide (25 mg) in methylene chloride (5 ml) was stirred for 4 hours at room temperature. The mixture was diluted with ethyl acetate and washed with dilute sodium carbonate solution. The ethyl acetate solution was then extracted twice with 0.1N hydrochloric acid and the combined acidic extracts were treated with enough 10% by weight sodium hydroxide solution to adjust the pH to 8.0. The solution was extracted twice with ethyl acetate and the combined ethyl acetate extracts were washed with brine, dried (anhydrous sodium sulfate) and evaporated to give a colorless oil (36 mg).

c) Synthesis of glutamic acid-y- (03-amidoethylmorphine) -a-benzyl ester-trifluoroacetate

A solution of N-t-BOC-glutamic acid morphine derivative (36 mg, prepared as described above) in methylene chloride (3 ml) was stirred at room temperature and trifluoroacetic acid (1 ml) was added. The mixture was stirred for 15 minutes and then concentrated to dryness. The residue was a colorless glass (40 mg).

d) Synthesis of methotrexate-y- (03-amidoethylmorphine) -a-benzyl ester

A solution of 4-amino-4-deoxy-N10-methylpteroic acid (37 mg) in 3 ml of dimethyl sulfoxide (DMSO), stirred at room temperature, was treated with triethylamine (28 μl) and i-butyl chloroformate (25 (il), and the mixture was stirred for 30 minutes, then it was added to a mixture of the morphine derivative (75 mg, prepared as described under c) and triethylamine (28 ml) in DMSO (2 ml), and the mixture was stirred at 60 ° for 1 hour touched. The cooled mixture was diluted with water and extracted twice with ethyl acetate. The combined ethyl acetate extracts were washed with water and then extracted twice with 0.1N hydrochloric acid. The combined acidic extracts were treated with enough 10 weight percent sodium hydroxide to raise the pH to 8.0. The mixture was extracted three times with ethyl acetate and the combined extracts were washed with brine (over anhydrous sodium sulfate), dried and concentrated to give a yellow oil (15 mg). This was purified by preparative thin layer chromatography on silica gel with development with chloroform / methanol, 4: 1. The desired compound was obtained as a yellow solid (4 mg).

e) Synthesis of metrotrexate-y- (03-amidoethylmorphine)

The product (4 mg) prepared as described under d) was mixed with 0.1N sodium hydroxide solution (5 ml) and the mixture was stirred at room temperature for 8 hours, resulting in a clear yellow solution.

0.1 N hydrochloric acid (5 ml) was added and the mixture was made up to 25 ml with sodium orthophosphate buffer (0.05 m, pH 7.4) to give a solution of the desired compound which is suitable for use in Enzyme test is suitable.

Example 7

Enzyme inhibitor immunoassay for morphine

A mixture of 10 jil morphine solution of suitable concentration, 50 (il methotrexate-y- (03-amidoethylmorphine) solution (4 ng / 25 ml), 50 jil antimorphine antibody solution, 10 | jl NAD PH solution, 10 jal 2 -Mercaptoethanol solution, 50 μl 1 potassium chloride solution, 150 μl Tris-HCl buffer solution (pH 7.5) with ÄDTA and 10 μl dihydrofolate reductase (E. casei) solution was incubated for 20 min at 37 ° C. The enzyme activity

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in the mixture was determined after adding 10 µl of dihydrofolate solution by recording the change in absorbance at 340 nm using a Centrifichem centrifugal analyzer. The results are shown in Table III below:

Table III

Reaction components enzyme activity

Morphine Antimor- Morphin Enzyme- OD / min% -Hem-

tig / sample phin metho test

Antibody trexate compo

by conjugate

0

missing missing

existing

0.54

0

the

0

is missing

3x 10 "1

! m existing

0.14

76

the

0

existing

3x 10 "1

! m existing

0.41

29

the

the

0.04

existing

3x10- '

! m existing

0.37

36

the

the

0.04

existing

3 x 10 ~!

! m existing

0.35

40

the

the

4.0

existing

3x IO "1

sm existing

0.31

46

the

the

20th

existing

3 x 10 "'

! m existing

0.27

54

the

the

40

existing

3x10- '

! m existing

0.23

60

the

the

10th

20th

25th

The table above shows that the activity of dihydrofolate reductase decreases with increasing concentration of morphine. For example, morphine solutions with four µg / ml to 4 mg / ml morphine could be tested, whereby only 10 μl solution was available. However, this is not intended to mean that this is the required area, but only demonstrates the successful application.

Example 8

Synthesis of ferritin-methotrexate conjugate

A solution of ferritin from human liver (1.3 mg) in sodium phosphate buffer (0.05 m, pH 8.0.5 ml) and 1 ml of dimethylformamide (DMF) was stirred at room temperature and 100 ml of a solution was obtained by treating methotrexate (23 mg) in DMF (2 ml) with triethylamine (21 ml) and i-butyl chloroformate (15 p.1) at room temperature for 30 min were added. The mixture was stirred at room temperature for 2 h and then dialyzed against 2 × 21 sodium orthophosphate buffer (0.05 m, pH 7.4, 0.1 m sodium chloride and with 0.05% by weight sodium azide) for 24 h. The solution was then passed through a Sephadex G-25 column which had been equilibrated with the same buffer and the fractions containing ferritin were pooled and made up to 10 ml with the buffer.

The resulting solution is suitable for use in an enzyme immunoassay for the determination of ferritin.

Claims (28)

641 570 2nd PATENT CLAIMS 1. A method for determining an immunologically active material in a sample, characterized in that the sample with a receptor, which is able to bind the immunologically active material specifically, with the immunologically active material, which with a substance, the degree and the type is capable of modifying the activity of an enzyme, is labeled, and is brought together with an enzyme and an enzyme substrate, the degree or type of enzyme activity obtained being measured and compared with that obtained with a standard. 2. The method according to claim 1, characterized in that the immunologically active material is brought together in an insolubilized form with an enzyme and an enzyme substrate, the degree or type of enzyme activity being measured after the separation of the solid phase and with that obtained with a standard is compared. 3. The method according to claim 1 or 2, characterized in that an enzyme inhibitor is used as a substance capable of modifying the activity of an enzyme. 4. The method according to any one of claims 1 to 3, characterized in that the receptor is an antibody. 5. The method according to any one of claims 1 to 4, characterized in that an inhibitor of the enzyme dihydrofolate reductase is used as an inhibitor. 6. The method according to any one of claims 1 to 5, characterized in that methotrexate is used as the inhibitor and dihydrofolate reductase is used as the enzyme. 7. The method according to any one of claims 1 to 6, characterized in that a hapten is used as the immunologically active material. 8. The method according to claim 7, characterized in that an digoxin antibody is used as the hapten digoxin and as a receptor. 9. The method according to claim 7, characterized in that an antibody to morphine is used as the hapten morphine and as the receptor. 10. The method according to any one of claims 1 to 6, characterized in that a protein is used as the immunologically active material. 11. The method according to claim 10, characterized in that human serum albumin is used as the protein and an antibody to human serum albumin is used as the receptor. 12. Reagent for carrying out the method according to claim 1 or 2, containing the immunologically active material or a receptor which is capable of specifically binding the immunologically active material, labeled with a substance which is capable of modifying the degree or type of activity of an enzyme . 13. A reagent according to claim 12, the immunologically active material of which is labeled with a substance capable of modifying the degree or type of activity of an enzyme. 14. A reagent according to claim 12, the receptor which is capable of specifically binding the immunologically active material is labeled with a substance which is capable of modifying the degree or type of activity of an enzyme. 15. Reagent according to claim 14, the receptor of which is an antibody specific for the immunologically active material to be determined. 16. Reagent according to one of claims 12 to 14, the substance capable of modifying the activity of an enzyme is an enzyme inhibitor. 17. A reagent according to claim 16, whose enzyme inhibitor has an inhibition constant below 10-3 mol / 1. 18. The reagent of claim 17, wherein the enzyme inhibitor has an inhibition constant between 10 ~ 15 and 10 ~ 15 mol / 1. 19. The reagent of claim 18, its inhibitor 5 Methotrexate and its enzyme is dihydrofolate reductase. 20. Reagent according to one of claims 12 to 15, the substance capable of modifying the activity of an enzyme is an enzyme activator. 21. Reagent according to one of claims 12 to 20, the i ° immunologically active material is digoxin. 22. A reagent according to any one of claims 12 to 20, the immunologically active material of which is human serum albumin. 23. A reagent according to any one of claims 12 to 20, the '5 immunologically active material is morphine. 24. Reagent according to one of claims 12 to 20, the immunologically active material of which is ferritin. 25. Reagent according to one of claims 12 to 24, its immunologically active material or its receptor, the 20 is able to specifically bind the immunologically active material, is covalently bound to the substance that is able to modify the degree or type of activity of an enzyme. 26. Reagent according to claim 21, consisting of a conjugate of digoxin and methotrexate. 25 27. Reagent according to claim 22, consisting of a conjugate of human serum albumin and methotrexate. 28. Reagent according to claim 23, consisting of a conjugate of morphine and methotrexate. 29. Reagent according to claim 24, consisting of a conjugate of ferritin and methotrexate.